Exploring the Potential of Cytochrome P450 CYP109B1 Catalyzed Regio-and Stereoselective Steroid Hydroxylation - Frontiers
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ORIGINAL RESEARCH
published: 18 February 2021
doi: 10.3389/fchem.2021.649000
Exploring the Potential of Cytochrome
P450 CYP109B1 Catalyzed
Regio—and Stereoselective Steroid
Hydroxylation
Xiaodong Zhang 1†, Yun Hu 1†, Wei Peng 2†, Chenghua Gao 1, Qiong Xing 1*, Binju Wang 2* and
Aitao Li 1*
1
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation
of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, China,
2
State Key Laboratory of Physical Chemistry of Solid Surfaces, Collaborative Innovation Center of Chemistry for Energy Materials,
National Engineering Laboratory for Green Chemical Productions of Alcohols, Ethers and Esters, College of Chemistry and
Chemical Engineering, Xiamen University, Xiamen, China
Edited by: Cytochrome P450 enzyme CYP109B1 is a versatile biocatalyst exhibiting hydroxylation
Gao-Wei Zheng,
activities toward various substrates. However, the regio- and stereoselective steroid
East China University of Science and
Technology, China hydroxylation by CYP109B1 is far less explored. In this study, the oxidizing activity of
Reviewed by: CYP109B1 is reconstituted by coupling redox pairs from different sources, or by fusing it to
Jian-bo Wang, the reductase domain of two self-sufficient P450 enzymes P450RhF and P450BM3 to
Hunan Normal University, China
Wuyuan Zhang,
generate the fused enzyme. The recombinant Escherichia coli expressing necessary
Xi’an Jiaotong University, Xi’an, China proteins are individually constructed and compared in steroid hydroxylation. The
*Correspondence: ferredoxin reductase (Fdr_0978) and ferredoxin (Fdx_1499) from Synechococcus
Qiong Xing
elongates is found to be the best redox pair for CYP109B1, which gives above 99%
qiongxingnmr@hubu.edu.cn
Binju Wang conversion with 73% 15β selectivity for testosterone. By contrast, the rest ones and the
wangbinju2018@xmu.edu.cn fused enzymes show much less or negligible activity. With the aid of redox pair of
Aitao Li
aitaoli@hubu.edu.cn
Fdr_0978/Fdx_1499, CYP109B1 is used for hydroxylating different steroids. The
†
These authors have contributed
results show that CYP109B1 displayed good to excellent activity and selectivity toward
equally to this work four testosterone derivatives, giving all 15β-hydroxylated steroids as main products except
for 9 (10)-dehydronandrolone, for which the selectivity is shifted to 16β. While for
Specialty section:
substrates bearing bulky substitutions at C17 position, the activity is essentially lost.
This article was submitted to
Catalysis and Photocatalysis, Finally, the origin of activity and selectivity for CYP109B1 catalyzed steroid hydroxylation is
a section of the journal revealed by computational analysis, thus providing theoretical basis for directed evolution
Frontiers in Chemistry
to further improve its catalytic properties.
Received: 03 January 2021
Accepted: 18 January 2021 Keywords: cytochrome P450, steroids hydroxylation, regioselectivity, stereoselectivity, redox partner, CYP109B1
Published: 18 February 2021
Citation:
Zhang X, Hu Y, Peng W, Gao C, INTRODUCTION
Xing Q, Wang B and Li A (2021)
Exploring the Potential of Cytochrome
Cytochrome P450 monooxygenases (CYPs) are heme-containing monooxygenase enzymes, which
P450 CYP109B1 Catalyzed
Regio—and Stereoselective
are broadly distributed among biological kingdoms and extensively involved in natural product
Steroid Hydroxylation. biosynthesis, degradation of xenobiotics, steroid biosynthesis, drug metabolism etc. (Munro et al.,
Front. Chem. 9:649000. 2007; Bernhardt and Urlacher, 2014; Li et al., 2020). They are considered to be the most versatile
doi: 10.3389/fchem.2021.649000 biocatalysts with the capability of catalyzing functionalization of non-activated hydrocarbons under
Frontiers in Chemistry | www.frontiersin.org 1 February 2021 | Volume 9 | Article 649000
Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
mild reaction conditions in a regio- and stereoselective manner, thus MATERIALS AND METHODS
accomplishing the challenging reactions that are difficult to be
achieved in chemical approach (Nelson, 2018; Wang et al., 2020; Materials
Zhang et al., 2020b). Due to the above reasons, CYPs have attracted Tryptone and yeast extract were purchased from OXOID
more and more attention with many CYPs discovered, identified, (Shanghai, China), isopropyl β-D-1-thiogalactopyranoside
characterized, and investigated for many types of oxidations of a vast (IPTG, >99%) and kanamycin sulfate (>99%) were bought
number of substrates, including hydroxylation (Guengerich, 2003; from Sangon (Shanghai, China) and steroid compounds were
Lewis et al., 2011; Li et al., 2013; Li et al., 2015; Li et al., 2016a; Prier purchased from Aladdin (Shanghai, China). All chemicals were of
et al., 2017), alcohol oxidation, N-oxidation (Wang et al., 2014), N-, chemical purity and commercially available. Prime STAR Max
O-, S-dealkylation (Mallinson et al., 2018), and C-C bond cleavage DNA polymerase was bought from Takara (Shanghai, China), T5
(Urlacher and Girhard, 2019), as well as unusual reactions such as super PCR Mix (Colony) DNA polymerase, DNA Maker,
nitration of tryptophan, cyclopropanation (Coelho et al., 2013), and
intramolecular C-H amination (Bernhardt et al., 2006; Isin and
™
Trelidf Prestained Protein Ladder and TS-GelRed Nucleic
acid dye were purchased from TSINGKE (Beijing, China). T5
Guengerich, 2007). exonuclease was obtained from New England Biolabs
Steroid-based drugs are the second largest marketed drugs (Beverley, MA).
after antibiotics, and CYPs catalyzed steroid hydroxylation is very
important in pharmaceutical application due to the enhanced
biological activity by this particular modification (Li et al., 2002; Construction of Recombinant E. coli Cells
Bureik and Bernhardt, 2007; Donova, 2017). So far, a number of as Whole-Cell Catalysts
CYPs have been isolated from different origins (plants, animals Full length gene of CYP109B1 was amplified from the genome
and microorganisms) for steroid hydroxylation (Bureik and of Bacillus subtilis 168 (Kunst et al., 1997; Zhang et al., 2020)
Bernhardt, 2007). Among them, microbial CYPs shows great using specific upstream and downstream primers by
superiority over the eukaryotic ones (Donova and Egorova, 2012; polymerase chain reaction (PCR) (Supplementary Table
Li et al., 2020a). Because they can be overexpressed in high S1), the resulted PCR fragment was ligated into the
amounts in soluble form (Bernhardt and Urlacher, 2014) and expression vector pRSFDuet-1 under control of T7 promoter
are generally much more active with turnover numbers go from using T5 exonuclease-dependent assembly approach (Xia et al.,
ten to a few hundred molecules per min. Therefore, more and 2019). The plasmid pRSFDuet-1-CYP109B1 was transformed
more CYPs from different bacterial species capable of into E. coli BL21 (DE3) for protein expression and further
hydroxylating steroids with different regio- and characteristic assay.
stereoselectivity have been investigated, such as P450 families In order to investigate the effect of different redox partners on
of CYP106, CYP154, CYP260, and CYP109 (Agematu et al., 2006; the catalytic efficiency of CYP109B1, several pairs of redox
Arisawa and Agematu, 2007; Zhang et al., 2020a). Moreover, partners from different sources were screened and optimized.
most of them have been employed and subjected to directed First, the ferredoxin reductase Fdr_0978 and the ferredoxin
evolution to improve the both activity and selectivity for steroid Fdx_1499 from Synechococcus elongates PCC7942 (Plas et al.,
hyxroxylation. 1988; Sun et al., 2016) was linked with RBS via overlap PCR
Nevertheless, for the enzyme P450 CYP109B1 from CYP109 and integrally inserted into the second multiple cloning site
family, although it has been reported many times for catalyzing (MCS2) of pRSFDuet-1-CYP109B1 followed by transformed
broad spectrum substrates including fatty acids, primary into E. coli BL21 (DE3). In the same way, ferredoxin reductase
n-alcohols and terpenoids, the use of it for steroid FNR and ferredoxin Fd I from spinach (Aliverti et al., 1995;
hydroxylation is far less explored (Furuya et al., 2008; Girhard Binda et al., 1998; Mulo and Medina, 2017), and the ferredoxin
et al., 2009; Girhard et al., 2010). And it was only used for reductase Fpr from E. coli (Bakkes et al., 2015; Bakkes et al.,
testosterone hydroxylation to give 15β hydroxylated product in 2017) grouped with ferredoxin YkuN and ferredoxin YkuP
the presence of truncated adrenodoxin reductase (AdR) and from B. subtilis (Girhard et al., 2010), respectively, were also
adrenodoxin (Adx) from bovine adrenocortical mitochondria, constructed for in vivo steroid hydroxylation. In addition, the
but with very low activity (5%–10% conversion). plasmids pRSFDuet-1-CYP109B1-RhF and pRSFDuet-1-
In this study, the potential of CYP109B1 catalyzed steroid CYP109B1-BM3 carrying the genes of fused enzymes were
hydroxylation was further explored. Frist, several pairs of electron constructed by fusing the heme domain of CYP109B1 to the
transfer partners from different sources were employed and N-terminal of reductase domain of P450RhF from
compared for achieving the best activity reproduction. Then, Rhodococcus sp. Strain NCIMB 9784 and P450BM3 from B.
different substrates including four testosterone derivatives and megatherium, respectively.
four steroids bearing bulky substitutions at C17 position were To express CYP109B1 and redox partners, a 5 mL
catalyzed by CYP109B1 to explore its substrate scope. Finally, the preculture of recombinant E. coli (DE3) cells was grown
origin of regio- and stereoselectivity of CYP109B1 catalyzed overnight in LB medium containing 50 mg/mL kanamycin
hydroxylation for different steroids was revealed, thus sulfate at 37°C (220 rpm). The seed culture was used to
providing basis of further directed evolution on CYP109B1 to inoculate a 200 mL Terrific Broth (TB) medium in a 500 mL
improve both activity and selectivity for practical industrial baffled flask at 37°C and 220 rpm. When the optical density
application. (OD600) reached 0.6, Isopropyl-thio-β-D-galactopyranoside
Frontiers in Chemistry | www.frontiersin.org 2 February 2021 | Volume 9 | Article 649000
Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
(IPTG) was added to give a final concentration of 0.2 mM for Kinetic Analysis of CYP109B1 Catalyzed
protein induction at 25°C and 200 rpm for 14 h. Cells were Steroid Hydroxylation
harvested by centrifugation at 4,000 rpm for 10 min and To determine the kinetic parameters of CYP109B1 catalyzed
thallus was washed twice with potassium phosphate buffer steroid hydroxylation, reactions were performed in 250 μL
(pH 8.0), then resuspended in the same buffer controlling sodium phosphate buffer (pH 8.0,100 mM) containing 1 μM
OD600 around 20 followed by quick-frozen in liquid nitrogen CYP109B1, 4 μM Fdr_0978, 20 μM Fdx_1499, 5% glycerol, 5%
until further use. glucose, 1 U 6-glucase-phosphate-dehydrogenase and 2 mM
MgCl2, at 30°C, 750 rpm for 2 min. The final substrate
concentration was ranged from 0 mM to 1.5 mM. And the
Steroid Hydroxylation With Recombinant
reaction was started by the addition of NADPH to a final
Whole-Cell Catalysts concentration of 1 mM followed by shaking at 30°C, 750 rpm
The reaction mixtures consist of 5 mL cell suspension in for 5 min, and quenched with an equivalent volume of
potassium phosphate buffer (100 mM, pH 8.0) containing acetonitrile. The substrate/production formation rate was
1 mM steroid substrates (1-8 dissolved in dimethyl determined by HPLC analysis. The values of Vmax, Km, and
formamide), 5% (v/v) glucose, 1 U 6-glucose-phosphate- kcat were determined by plotting the substrate consumption
dehydrogenase and 5% (v/v) glycerol. The reactions were rate vs. the corresponding substrate concentration using a
initiated by the addition of 1 mM NADP+ at 25°C, 200 rpm hyperbolic fit in GraphPad Prism 8.0 software (La Jolla, CA,
for 9 h. After reaction, 500 μL reaction mixture was taken and United States). And the kinetic parameters are displayed in
the products were extracted with 500 μL ethyl acetate, the organic Table 1 and Supplementary Figure S3.
phase was obtained by centrifugation at 12,000 rpm, which was For the determination of coupling efficiency of CYP109B1 for
then evaporated before resuspended in an equal volume different steroid substrates, reaction mixture containing 1 μM
acetonitrile for HPLC analysis. CYP109B1, 4 μM Fdr_0978, and 20 μM Fdx_1499, 1 mM
substrate and 2 mM NADPH in potassium phosphate buffer
(100 mM, pH 8.0). And reaction was performed at 30°C,
Expression and Purification of CYP109B1
750 rpm for 6 min. Consumption of NADPH was measured
and Corresponding Redox Partners by absorbance variation at 340 nm with UV-1800
All proteins fused to a N-terminal poly histidine tag were Spectrophotometer and product formation was analyzed via
overexpressed in E. coli BL21 (DE3) cell and purified using HPLC by addition of equivalent volume of acetonitrile. The
Ni-NTA column. For the purification of CYP109B1, E. coli coupling efficiency was calculated by the ratio of the product
BL21 (DE3) strain harboring pRSFDuet-1-CYP109B1, was concentration with consumption concentration of NADPH
inoculated as described above. And cells were harvested by (Table 1).
centrifugation and resuspended in 50 mM potassium
phosphate buffer (pH 8.0) containing 500 mM NaCl and
10 mM imidazole, then disrupted by sonication on ice. The HPLC Analysis
crude cell extract was prepared by removal of cell debris by The conversion analysis of steroids and corresponding products
centrifugation at 9,000 rpm for 30 min and the supernatant was performed via reversed phase HPLC technique using a
was filtered through 0.45 μm pore size filters. The resulting Shimadzu LC2030C system equipped with an Agilent
cell-free extract was loaded onto an immobilized metal ion ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm; Agilent
affinity chromatography column that had been equilibrated Technologies, Santa Clara, CA, United States). CYP109B1
with 50 mM potassium phosphate buffer (pH 8.0) containing reaction mixtures were extracted with ethyl acetate and
500 mM NaCl. The column was washed with 10 column organic phase evaporates naturally followed by added equal
volumes of 50 mM potassium phosphate buffer (pH 8.0) volume chromatographic grade acetonitrile and then subjected
containing 500 mM NaCl and 20 mM imidazole in order to to HPLC analyze. The steroids and corresponding products were
remove non-specifically bound protein. Subsequently, the eluted using a gradient method (Supplementary Table S3),
tagged protein was eluted with 50 mM potassium starting with a mobile phase consisting of methanol,
phosphate buffer (pH 8.0) containing 500 mM NaCl, acetonitrile and water in a ratio of 15:15:70 with a flow rate of
100 mM imidazole. The concentration of CYP109B1 was 1.5 mL/min, injection volume of 10 μL and a temperature at 40°C.
examined by the CO difference spectrum analysis using an
extinction coefficient of 91 mM−1 cm−1 (Supplementary
Table S2; Supplementary Figure S2). Analogously, the Preparation and Identification of
expression and purification of redox partners were similar Hydroxylated Products by CYP109B1
as described above. And the concentrations of redox partners For preparation and identification of hydroxylated products from
were calculated by Bradford assay, using bovine serum CYP109B1 catalzyed steroid hydroxylation, large-scale
albumin as a standard. Protein purity and molecular cultivation 1 L Terrific Broth (TB) medium in a 2 L baffled
weight was assessed by SDS-PAGE. All purified proteins flask was carried out as described above. The reaction mixture
were stored in −80 °C after quick-frozen with liquid of l L was incubated at 25°C 200 rpm for 10 h with pH adjustment
nitrogen for further assays. (pH 8.0) during the reaction. After reaction, equal volume ethyl
Frontiers in Chemistry | www.frontiersin.org 3 February 2021 | Volume 9 | Article 649000
Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
TABLE 1 | Kinetic parameters of CYP109B1 catalyzed steroids hydroxylationa.
Substrate Km (μM) kcat (min−1) kcat/Km (M−1 min−1) NADPH consumption Coupling efficiency
rate (μmol/μmol*min) (%)b
1 83.7 ± 20.4 4.7 ± 0.3 5.6 × 104 74 ± 4.8 11 ± 5.3
2 136.3 ± 18.4 4.3 ± 0.1 3.1 × 104 48 ± 11.8 10 ± 1.5
3 193.1 ± 38.2 3.5 ± 0.2 1.8 × 104 147 ± 34.3 4 ± 0.9
4 163.1 ± 38.4 11.8 ± 0.7 7.2 × 104 67 ± 2.9 5 ± 2.7
a
Reaction conditions: Kinetic parameters: 1 μM enzyme, steroids (0–1.5 mM), NADPH regeneration system (5 g/L glucose-6-phosphate, 5 g/L glycerol, 1 unit glucose-6-phosphate
dehydrogenase and 1 mM NADPH). Reactions were allowed to proceed in 5 min with shaking at 750 rpm at 30°C. NADPH coupling efficiency: 1 μM mixture enzyme, 1 mM testosterone,
2 mM NADPH. Reaction mixtures were incubated at 750 rpm at 30°C for 6 min.
b
Coupling efficiency was calculated as the amount of the product produced divided by the amount of NADPH consumed.
acetate was then added for products extraction, followed by relaxation time of 0.001 ns Further equilibration of the systems
centrifugation to obtain the organic phase which was was allowed for 4 ns to get well settled temperature and
evaporated to give the crude products. The crude products pressure. After proper minimizations and equilibrations, a
were carefully loaded on a silica gel column (200–300 mesh productive MD run of 50 ns was performed for all the
particle size, 3.0 cm × 60 cm) with dichloromethane and complex systems.
methanol as elution solvent. Using gradient elution, the ratio
of methanol to dichloromethane gradually changed from 1:100 to NMR Characterization of the Products
1:50, the fractions containing the product were combined and All Nuclear Magnetic Resonance (NMR) spectra were recorded
dried in a rotary vacuum system to obtain the pure product that on Agilent 400 MHz spectrometer equipped with a 1H-19F/15N-
was used for NMR characterization for structural identification. 31P probe at 25°C. Samples (20 mg/mL) for NMR experiment
were typically prepared in dimethyl sulfoxide (DMSO) or
Deuterium generation of chloroform (CDCl) and NMR data
Molecular Dynamics (MD) Simulation and were processed and analyzed with MestReNova 14.2. Full 1H
Molecular Docking and 13C assignments, including the stereospecific assignment
The initial CYP109B1 structure was taken from protein data bank of prochiral 1H were obtained based on NMR spectra from
(PDB ID: 4RM4) (Zhang et al., 2015). The steroid substrates were standard 1D experiments as well as 1H DQF-COSY,
1
docked into the active site of heme domain using Auto-Dock H,1H-NOESY and 1H,13C-HSQC correlation experiments.
Vina in Discovery Studio based on a pose of CYP109B1 after a The 1H and 13C chemical shifts were further confirmed with
brief molecular dynamic (MD) simulation. The MD simulations the values from previous studies (Reetz et al., 2008; de Flines
of CYP109B1-progesteron and CYP109B1-canrenone complexes et al., 2015; Qiao et al., 2017).
were performed with GPU version of Amber 18 package (Case
et al., 2018). Missing hydrogen atoms were added by module leap
of Amber 18 (Case et al., 2018). The force field for the resting- RESULTS
state species were parameterized using the “MCPB.py” modeling
tool (Li et al., 2016b). The general AMBER force field (GAFF) Expression, Purification and Spectral
(Wang et al., 2004) was used for steroid substrates, while the Characterization of CYP109B1
partial atomic charges and missing parameters were obtained The recombinant expression of P450 CYP109B1 was performed
from the RESP method (Bayly et al., 1993), using HF/6–31G* in E. coli (BL21) as a soluble protein. Under the induction of
level of theory. 18 Na+ ions were added into the protein surface to 0.2 mM Isopropyl-thio-β-D-galactopyranoside (IPTG) for 14 h at
neutralize the total charges of the systems. Finally, the 25°C, the thallus of CYP109B1 displayed typical brick red color of
resulting systems were solvated in a cubic box of TIP3P cytochrome P450 monooxygenase (Supplementary Figure S1).
(Jorgensen et al., 1983) waters extending up to minimum The protein purification of CYP109B1 was conducted by
cutoff of 15 Å from the protein boundary. The Amber immobilized metal (Nickel) affinity chromatography with 6 ×
ff14SB force field (Maier et al., 2015) was employed for the His tag, and the purified protein of CYP109B1 with deeper brick
protein in all of the MD simulations. The initial structures red color was obtained. The theoretical protein size of CYP109B1
were fully minimized using combined steepest descent and is approximately 43.5 kDa with 396 gene-encoded residues,
conjugate gradient method. The systems were then gently which is in accordance with practical size (Supplementary
annealed from 10 to 300 K under canonical ensemble for Figure S1). Protein concentration of the purified protein was
0.05 ns with a weak restraint of 15 kcal/mol/Å. 1 ns of estimated to be 172 μΜ based on dithionite reduced and CO-
density equilibration were performed under isothermal- difference spectral assay with extinction coefficient of ε450–490
isobaric ensemble at target temperature of 300 K and the 91 mM−1 cm−1 (Omura and Sato, 1964), and a typical peak at
target pressure of 1.0 atm using Langevin-thermostat 450 nm as one of the major spectral characteristics of cytochrome
(Izaguirre et al., 2001) and Berendsen barostat (Berendsen P450 monooxygenase was also observed (Supplementary
et al., 1984) with collision frequency of 0.002 ns and pressure- Figure S2).
Frontiers in Chemistry | www.frontiersin.org 4 February 2021 | Volume 9 | Article 649000Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
FIGURE 1 | (A): The configuration of plasmids containing CYP109B1 with different pairs of redox partners (ferredoxin and ferredoxin reductase) or plasmids
containing fused enzyme by fusing the heme domain of CYP109B1 to reductase domain of P450 BM3 or P450RhF. a: plasmid pRSFDuet-1 containing CYP109B1 and
Fdr_0978/Fdx_1499 redox partner, b: plasmid pRSFDuet-1 containing CYP109B1 and FNR/Fd I redox partner from spinach, c: plasmid pRSFDuet-1 containing
CYP109B1 and Fpr/YkuN, d: plasmid pRSFDuet-1 containing CYP109B1 and Fpr/YkuP, e: plasmid pRSFDuet-1 containing fused enzyme P450CYP109B1-BM3
in which heme domain of CYP109B1 was fused to the reductase domain of P450 BM3 from Bacillus megaterium, f: plasmid pRSFDuet-1 containing fused enzyme
P450CYP109B1-RhFRed in which heme domain of CYP109B1 was fused to the reductase domain of P450RhF from Rhodococcus sp. Strain NCIMB 9784. (B): SDS-
PAGE analysis of recombination E. coli cells containing the corresponding plasmids in Figure 1A. (C): Conversion of testosterone with whole-cell catalyst expressing
different reconstituted catalytic system. Reaction conditions: recombinant E. coli cells frozen with liquid nitrogen was suspended in phosphate buffer (pH 8.0, 100 mM,
OD600 20) containing 1 unit glucose-6-phosphate dehydrogenase, 5 g/L glucose-6-phosphate, 5 g/L glycerol and 1 mM NADP+, reactions were conducted at 25°C,
200 rpm for 20 h.
Reconstitution of P450 CYP109B1 Activity CYP109B1 to the reductase domain of P450RhF from
for Steroid Hydroxylation Rhodococcus sp. Strain NCIMB 9784 or P450BM3 from B.
In order to explore the potential of P450 CYP109B1 for steroid megatherium (Figure 1A, entries f and g).
hydroxylation in drug synthesis, the electron transfer proteins Next, the plasmids harboring the corresponding genes were
flavodoxin reductase (Fpr) from Escherichia coli, flavodoxins individually transformed into recombinant E. coli, which resulted
in six different whole-cell catalysts. The SDS-PAGE analysis was
(YkuN or YkuP) from B. subtilis (Girhard et al., 2010),
then performed to check the protein expression for either three-
ferredoxin reductase (Fdr_0978) and ferredoxin (Fdx_1499)
component catalytic system or self-sufficient catalytic system. As
from Synechococcus elongates PCC7942 (Plas et al., 1988; Sun
shown in Figure 1B, all the proteins were successfully expressed
et al., 2016) as well as ferredoxin reductase (FNR) and
including the fused P450 enzymes. Subsequently, the whole-cell
ferredoxin (Fd I) from spinach (Aliverti et al., 1995; Binda as catalysts were compared for steroid hydroxylation using
et al., 1998; Mulo and Medina, 2017) were employed for the testosterone as the model substrate. The results are presented
reconstitution of activity of CYP109B1. Based on different in Figure 1C, the reconstituted catalytic system with CYP109B1
combination, four pairs of redox partners Fdr_0978/ coupled to the redox pair of Fdr_0978/Fdx_1499 showed the best
Fdx_1499, Fpr/YkuN, Fpr/YkuP and FNR/FdI were tested catalytic activity and 99% conversion was achieved to give 15β-
for their performance in CYP109B1catalyzed testosterone hydroxylated product as main product with 78% selectivity
hydroxylation (Figure 1A, entries a-d). In addition, two (Supplementary Figure S4). In addition, when coupled with
chimeric proteins were also constructed by fusing the another two pairs of redox partners Fpr/ YkuN and Fpr/YkuP,
Frontiers in Chemistry | www.frontiersin.org 5 February 2021 | Volume 9 | Article 649000Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
obvious activity was also observed with substrate conversion position (55%) vs. 15β-hydroxylated product (29%) due to the
being 49% and 60%, respectively (Supplementary Figure S4). different spatial structure of the substrate 4 (Supplementary
One possible reason could be the lower protein expression of Fpr/ Figure S8) (Supplementary Figures S17, S18). Obviously,
YkuN and Fpr/YkuP compared with Fdr_0978/Fdx_1499 C10 position of steroids 2 and 4 lack an angular methyl group
(Figure 1C). Although the natural redox partners of comparted to 1 and 3, and molecular structural difference might
CYP109B1 have not been discovered, we have successfully lead to the decreased or switched selectivity. On the other hand,
identified a pair of redox partner ferredoxin reductase for those substrates with extra side substitution at C17 such as
Fdr_0978 and ferredoxin Fdx_1499 from Synechococcus progesterone (5), canrenone (6), prednisolone (7) and
elongates PCC7942 which could efficiently deliver electrons pregnenolone (8), which are strictly rejected from active
from NADPH to CYP109B1 for steroid hydroxylation. pocket of CYP109B1 and negligible activity was observed
On the other hand, the two fused enzymes exhibited negligible (Supplementary Figures S9-S12).
activity, although proteins were well expressed. The result It was known that the corresponding product are interesting
indicated that P450CYP109B1 is incompatible with the pharmaceutical intermediates for the production of anti-
reductase domain of the self-sufficient P450RhFed and inflammatory, diuretic, anabolic, contraceptive, antiandrogenic,
P450BM3 for the activity reproduction. In summary, with the progestational, and antitumor drugs, or they are themselves
support of heterogeneous redox partner ferredoxin reductase biologically active (Fernandes et al., 2003; Furuya et al., 2009;
(Fdr_0978) and ferredoxin (Fdx_1499) from Synechococcus Donova and Egorova, 2012). To the best of our knowledge, some
elongates PCC7942, P450 CYP109B1 showed excellent catalytic of the steroidal C15 or C16 alcohols have not been reported to
activity for steroid hydroxylation. Therefore, the reconstituted date, for example, 15β-hydroxynandrolone, 15β-
catalytic system of CYP109B1-Fdr_Fdx was selected for hydroxyboldenone and 16β-hydroxy9 (10)dehydronandrolone.
further study. Thus, CYP109B1 has great potential as steroid hydroxylase and
used in the production of high value-added steroids drugs in
industrial application.
P450 CYP109B1 Catalyzed Hydroxylation of
Different Steroids
As a versatile P450 monooxygenase, P450CYP109B1 could accept KINETIC STUDY OF CYP109B1
various substrates including compactin, terpenoids like CATALYZED STEROID HYDROXYLATION
valencene, fatty acid and primary alcohols as good substrates
(Girhard et al., 2009; Zhang et al., 2015). For the steroid In order to make a further insight into the catalytic efficiency of
hydroxylation, it was only reported that testosterone (Girhard CYP109B1 for steroid hydroxylation, kinetics and NADPH
et al., 2010) could be catalyzed by CYP109B1 to form the 15β- coupling efficiency for substrates 1-4 were determined.
hydroxylated testosterone as main product in the presence of Measurements were carried out using pure P450CYP109B1
redox partners Adr/Adx from bovine adrenocortical together with purified redox partners Fdr_0978 and Fdx_1499,
mitochondria, but with very low activity (5–10% conversion). a molar ratio of CYP109B1: Fdr: Fdx 1: 4: 20 was employed and
Afterward, although more steroids like androstenedione and the results are listed in Table 1. Among the four substrates tested,
norethindrone have also been identified to be catalyzed by P450 CYP109B1 exhibited the best affinity toward testosterone 1
CYP109B1 (Furuya et al., 2008), but the corresponding and Km value was estimated to be approximately 84 μΜ. While
hydroxylated products have never been identified yet. for catalytic performance, the best catalytic performance (kcat/
Therefore, the constructed reconstituted catalytic system Km) was obtained for substrate 4, which was measured to be 7.2 ×
P450 CYP109B1-Fdr_Fdx with the highest catalytic efficiency 104 M−1 min−1, indicating that 9 (10)-dehydronandrolone was
were used in biotransformation of different steroid substrates. the most preferable steroid substrate for CYP109B1 in terms of
As shown in Figure 2, CYP109B1 showed excellent activity catalytic activity. In addition, coupling efficiency of CYP109B1
toward testosterone (1) and its derivatives nandrolone (2), for substrates 1-4 were also tested to further characterize the
boldenone (3) and 9 (10) dehydronandrolone (4). For performance of this enzymatic system. It was found that enzyme
substrates testosterone (1) and boldenone (3), good activity CYP109B1 showed relatively low coupling efficiency for all the
was also achieved with the 79% and 78% conversion, substrate and the highest value of 11 was achieved for
respectively. The main products produced was identified by testosterone.
HPLC/MS and NMR spectroscopy (data are listed in
Supplementary Material) (Supplementary Figures S25-S41).
In terms of selectivity, main hydroxylation occurs at 15β Mechanism Study on CYP109B1 Catalyzed
position for substrates 1 and 3, to give the 15β-hydroxylated Steroid Hydroxylation
product with above 70% selectivity (Supplementary Figures S5, To identify the possible binding conformations of steroids in
S7) (Supplementary Figures S13, S16). And for substrate 2, the active site of CYP109B1, substrates 1-4 were docked into the
selectivity decreased to 54% due to the formation of 16β- active pocket of CYP109B1 (Supplementary Figures S19-S23).
hydroxylated nandrolone (29%) as minor product The docking results showed that the C15 β-hydrogen of
(Supplementary Figure S6) (Supplementary Figures S14, testosterone (1) is well positioned for H-abstraction, with a
S15). By contrast, major hydroxylation happened at 16β distance of 2.0 Å between C-15 atom of 1 with the O atom of
Frontiers in Chemistry | www.frontiersin.org 6 February 2021 | Volume 9 | Article 649000Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
FIGURE 2 | Regio- and stereoselective hydroxylation with CYP109B for eight different steroid substrates: testosterone (1), nandrolone (2), boldenone (3), 9 (10)-
dehydronandrolone (4), progesterone (5), Canreone (6), Prednisolone (7) and Pregnenolone (8). Whole cell reaction of CYP109B1-Fdr_Fdx displayed catalyze activity for
substrate 1 to 4, and major products were listed.
Cpd I (Figure 3A). In addition, hydrophobic interactions were By contrast, the C15, C16 site in substrate 5 and 6 are quite
formed between substrate 1 with residues Ile82, Leu235 and far away from the heme-iron (Figure 4). As such, we
Ala239, Pro285 and Ala286 located in the active pocket of speculate that stable electrostatic interactions between Fe
CYP109B1. For the analogous substrate nandrolone (2), the and keto O atom may hinder the binding of initial O 2 co-
docked conformation is quite similar to that of 1, where C15 substrate, which in turn inhibit the formation of the active
carbon atom of 2 maintains a distance of 2.2Å with the O atom of specie Cpd I. Thus, the extra side substitution at C17 of sterol
Cpd I (Figure 3B), leading to the formation of 15β-hydroxylated seriously impedes the active binding of the O 2 co-substrate
steroid as main product. Compared with substrates 1 and 2, the which into active pocket of CYP109B1.
docked result of substrate boldenone (3) showed that additional
H-bonding interactions were formed between C17-hydroxyl of 3
and Gly240 and Thr243, C3-keto of 3 and Leu289 (Figure 3C), CONCLUSION
which may increase the binding affinity and stability of 3. All these
would in turn lead to the highest catalytic selectivity for substrate 3 In summary, we have established a more efficient CYP109B1
among those steroids. As for substrate 9 (10)-dehydronandrolone catalytic system for steroid hydroxylation by screening redox
(4), different from the three steroids mentionsed above, C16 carbon pairs from different resources or constructing the fused
atom of 4 keeps a short distance of 1.7 Å with the O atom of Cpd I. enzymes by fusing CYP109B1 to the N-terminal of
Along with the binding conformation of 4 (Figure 3D), the reaction reductase domain of self-sufficient P450 BM3 and
would lead to the switched selectivity to produce the 16β- P450RhF. The three-component system using the
hydroxylated steroid. Fdr_0978/Fdx_1499 as redox partner showed the highest
Unlike the active steroid substrates 1-4, the substrates catalytic activity. Based on it, the substrate scope was
progesterone (5), canrenone (6), prednisolone (7) and tested for CYP109B1 and the subsequent computational
pregnenolone (8) carrying the extra side chain at C17 were analysis was performed , which enabled us to reveal the
found to be inactive in experiments. To understand the root origin of regio- and stereoselective steroid hydroxylation of
cause for this, the substrates 5 and 6 were further docked into different steroids substrates. Future work will be focusing on
the pocket of CYP109B1 in the resting state. As can be seen in the engineering of CYP109B1 to further improve the activity
Supplementary Figure S24, the O atom of keto at the A ring or selectivity for steroid hydroxylation, thus expanding its
of substrate 5 and the E ring of substrate 6 was found to point application in challenging biotransformation.
toward the heme-iron center, with a short distance of 1.8 Å
and 2.1 Å, respectively. Further MD simulations indicated
that such binding conformations of substrate 5 and 6 are ACKNOWLEDGMENTS
quite stable during 50 ns-MD simulation, during which the
O atom of keto in substrate 5 and 6 maintains an average We thank Shengying Li of Shandong University provide
distance of 2.3 Å and 2.6 Å with the heme-iron, respectively. the ferredoxin reductase Fdr_0978 and ferredoxin
Frontiers in Chemistry | www.frontiersin.org 7 February 2021 | Volume 9 | Article 649000Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation FIGURE 3 | The docked conformation of steroid substrates in the active pocket of CYP109B1 (PDB ID: 4RM4). (A): Testosterone. (B): Nandrolone. (C): Boldenone. (D): 9 (10) dehydronandrolone .Key residues in active pocket of CYP109B1 are colored in violet and residues forming hydrogen bonds are labeled in blue. The important distance in the favorable docked poses are given in angstrom (Å). The C15 atom of steroid substrates are displayed in green with ball style and the Ox atom of Cpd I are displayed in red with ball style. FIGURE 4 | The fluctuation of the distance of Fe—O3 (red), Fe—C15 (orange) and Fe—C16 (blue) in CYP109B1-Progesterone systems (A) and CYP109B1- Progesterone systems (B) during the MD simulations. Fdx_1499 from Synechococcus elongates PCC7942. We are DATA AVAILABILITY STATEMENT also grateful to Xudong Qu of Shanghai Jiao Tong University offered the ferredoxin reductase FNR and ferredoxin Fd I The raw data supporting the conclusions of this article will be from spinach. made available by the authors, without undue reservation. Frontiers in Chemistry | www.frontiersin.org 8 February 2021 | Volume 9 | Article 649000
Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
AUTHOR CONTRIBUTIONS and Development Program of China (2019YFA0905000 and
2019YFA0906400), the Distinguished Young Scholars of Hubei
AL conceived and provide advice in this research article. XZ was Province (2020CFA072), and was Supported by the Innovation
involved carrying out the experiment, data reduction and drafted base for Introducing Talents of Discipline of Hubei Province
the manuscript; YH, WP, and CG performed the mechanism (2019BJH021) as well as the Research Program of State Key
study and analyzed the data; QX, BW, and AL analyzed the data; Laboratory of Biocatalysis and Enzyme Engineering.
All authors read, approved and modified the final manuscript.
SUPPLEMENTARY MATERIAL
FUNDING
The Supplementary Material for this article can be found online at:
This work was funded by the National Natural Science Foundation https://www.frontiersin.org/articles/10.3389/fchem.2021.649000/
of China (No. 21977026 & 21702052), the National Key Research full#supplementary-material.
hydroxylation. Recl. Trav. Chim. Pays-Bas 82 (2), 139–142. doi:10.1002/recl.
REFERENCE 19630820205
Donova, M. V. (2017). Steroid bioconversions. Methods Mol. Biol. 1645, 1–13.
Agematu, H., Matsumoto, N., Fujii, Y., Kabumoto, H., Doi, S., Machida, K., et al. doi:10.1007/978-1-4939-7183-1_1
(2006). Hydroxylation of testosterone by bacterial cytochromes P450 using the Donova, M. V., and Egorova, O. V. (2012). Microbial steroid transformations:
Escherichia coli expression system. Biosci. Biotechnol. Biochem. 70 (1), 307–311. current state and prospects. Appl. Microbiol. Biotechnol. 94 (6), 1423–1447.
doi:10.1271/bbb.70.307 doi:10.1007/s00253-012-4078-0
Aliverti, A., Bruns, C. M., Pandini, V. E., Karplus, P. A., Vanoni, M. A., Curti, B., Fernandes, P., Cruz, A., Angelova, B., Pinheiro, H. M., and Cabral, J. M. S. (2003).
et al. (1995). Involvement of serine 96 in the catalytic mechanism of ferredoxin- Microbial conversion of steroid compounds: recent developments. Enzym.
NADP+ reductase: structure--function relationship as studied by site-directed Microb. Technol. 32 (6), 688–705. doi:10.1016/S0141-0229(03)00029-2
mutagenesis and X-ray crystallography. Biochemistry 34 (26), 8371–8379. Furuya, T., Nishi, T., Shibata, D., Suzuki, H., Ohta, D., and Kino, K. (2008).
doi:10.1021/bi00026a019 Characterization of orphan monooxygenases by rapid substrate screening using
Arisawa, A., and Agematu, H. (2007). A modular approach to biotransformation FT-ICR mass spectrometry. Chem. Biol. 15 (6), 563–572. doi:10.1016/j.
using microbial cytochrome P450 monooxygenases. Modern Biooxidation, chembiol.2008.05.013
177–192. doi:10.1002/9783527611522 Furuya, T., Shibata, D., and Kino, K. (2009). Phylogenetic analysis of Bacillus P450
Bakkes, P. J., Biemann, S., Bokel, A., Eickholt, M., Girhard, M., and Urlacher, V. B. monooxygenases and evaluation of their activity towards steroids. Steroids 74
(2015). Design and improvement of artificial redox modules by molecular (12), 906–912. doi:10.1016/j.steroids.2009.06.005
fusion of flavodoxin and flavodoxin reductase from Escherichia coli. Sci. Rep. 5, Girhard, M., Klaus, T., Khatri, Y., Bernhardt, R., and Urlacher, V. B. (2010).
12158. doi:10.1038/srep12158 Characterization of the versatile monooxygenase CYP109B1 from Bacillus
Bakkes, P. J., Riehm, J. L., Sagadin, T., Rühlmann, A., Schubert, P., Biemann, S., subtilis. Appl. Microbiol. Biotechnol. 87 (2), 595–607. doi:10.1007/s00253-
et al. (2017). Engineering of versatile redox partner fusions that support 010-2472-z
monooxygenase activity of functionally diverse cytochrome p450s. Sci. Rep. Girhard, M., Machida, K., Itoh, M., Schmid, R. D., Arisawa, A., and Urlacher, V. B.
7 (1), 9570. doi:10.1038/s41598-017-10075-w (2009). Regioselective biooxidation of (+)-valencene by recombinant E. coli
Bayly, C. I., Cieplak, P., Cornell, W., and Kollman, P. A. (1993). A well-behaved expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system.
electrostatic potential based method using charge restraints for deriving atomic Microb. Cell. Fact. 8 (1), 36. doi:10.1186/1475-2859-8-36
charges: the RESP model. J. Phys. Chem. 97 (40), 10269–10280. doi:10.1021/ Guengerich, F. P. (2003). Cytochrome P450 oxidations in the generation of reactive
j100142a004 electrophiles: epoxidation and related reactions. Arch. Biochem. Biophys. 409
Berendsen, H. J. C., Postma, J. P. M., van Gunsteren, W. F., DiNola, A., and Haak, (1), 59–71. doi:10.1016/S0003-9861(02)00415-0
J. R. (1984). Molecular dynamics with coupling to an external bath. J. Chem. Isin, E. M., and Guengerich, F. P. (2007). Complex reactions catalyzed by
Phys. 81 (8), 3684–3690. doi:10.1063/1.448118 cytochrome p450 enzymes. Biochim. Biophys. Acta 1770 (3), 314–329.
Bernhardt, R. (2006). Cytochromes P450 as versatile biocatalysts. J. Biotechnol. 124 doi:10.1016/j.bbagen.2006.07.003
(1), 128–145. doi:10.1016/j.jbiotec.2006.01.026 Izaguirre, J. A., Catarello, D. P., Wozniak, J. M., and Skeel, R. D. (2001). Langevin
Bernhardt, R., and Urlacher, V. B. (2014). Cytochromes P450 as promising stabilization of molecular dynamics. J. Chem. Phys. 114 (5), 2090–2098. doi:10.
catalysts for biotechnological application: chances and limitations. Appl. 1063/1.1332996
Microbiol. Biotechnol. 98 (14), 6185–6203. doi:10.1007/s00253-014- Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L.
5767-7 (1983). Comparison of simple potential functions for simulating liquid water.
Binda, C., Coda, A., Aliverti, A., Zanetti, G., and Mattevi, A. (1998). Structure of the J. Chem. Phys. 79 (2), 926–935. doi:10.1063/1.445869
mutant E92K of [2Fe-2S] ferredoxin I from Spinacia oleracea at 1.7 A Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., et al.
resolution. Acta Crystallogr. D Biol. Crystallogr. 54 (6), 1353–1358. doi:10. 1997). The complete genome sequence of the gram-positive bacterium Bacillus
1107/S0907444998005137 subtilis. Nature 390 (6657), 249–256. doi:10.1038/36786
Bureik, M., and Bernhardt, R. (2007). Steroid hydroxylation: microbial steroid Lewis, J. C., Coelho, P. S., and Arnold, F. H. (2011). Enzymatic functionalization of
biotransformations using cytochrome P450 enzymes. Mod. Biooxidation 155, carbon-hydrogen bonds. Chem. Soc. Rev. 40 (4), 2003–2021. doi:10.1039/
155. doi:10.1002/9783527611522.ch6 c0cs00067a
Case, D. A., Ben-Shalom, I. Y., Brozell, S. R., Cerutti, D. S., Cheatham, T. E., III, Li, A., Acevedo-Rocha, C. G., D’Amore, L., Chen, J., Peng, Y., Garcia-Borràs, M.,
Cruzeiro, V. W. D., et al. (2018). Amber 2018. San Francisco, CA: University of et al. (2020a). Regio- and stereoselective steroid hydroxylation at C7 by
California. doi:10.13140/RG.2.2.36172.41606 cytochrome P450 monooxygenase mutants. Angew. Chem. Int. Ed., 59,
Coelho, P. S., Brustad, E. M., Kannan, A., and Arnold, F. H. (2013). Olefin 12499. doi:10.1002/anie.202003139
cyclopropanation via carbene transfer catalyzed by engineered cytochrome Li, R.-J., Zhang, Z., Acevedo-Rocha, C. G., Zhao, J., and Li, A. (2020b). Biosynthesis
P450 enzymes. Science 339 (6117), 307–310. doi:10.1126/science.1231434 of organic molecules via artificial cascade reactions based on cytochrome p450
de Flines, J., van der Waard, W. F., Mijs, W. J., van Dijck, L. A., and Szpilfogel, S. A. monooxygenases. Green Synth. Catal. 1 (1), 52–59. doi:10.1016/j.gresc.2020.
(2015). Microbiological conversion of 19-nortestosterone: (III) 12- 05.002
Frontiers in Chemistry | www.frontiersin.org 9 February 2021 | Volume 9 | Article 649000Zhang et al. CYP109B1 Catalyzed Selective Steroid Hydroxylation
Li, A., Liu, J., Pham, S. Q., and Li, Z. (2013). Engineered P450pyr monooxygenase Qiao, Y., Shen, Y., Huang, W., Wang, Y., Ren, J., Xia, T., et al. (2017). Biocatalyst-
for asymmetric epoxidation of alkenes with unique and high enantioselectivity. mediated production of 11,15-dihydroxy derivatives of androst-1,4-dien-3,17-
Chem. Commun. 49 (98), 11572. doi:10.1039/c3cc46675b dione. J. Biosci. Bioeng. 123 (6), 692–697. doi:10.1016/j.jbiosc.2017.01.008
Li, A., Wu, S., Adams, J. P., Snajdrova, R., and Li, Z. (2015). Asymmetric Reetz, M. T., Kahakeaw, D., and Lohmer, R. (2008). Addressing the numbers problem in
epoxidation of alkenes and benzylic hydroxylation with P450tol directed evolution. Chembiochem 9 (11), 1797–1804. doi:10.1002/cbic.200800298
monooxygenase from Rhodococcus coprophilus TC-2. Chem. Commun. 50 Sun, Y., Ma, L., Han, D., Du, L., Qi, F., Zhang, W., et al. (2016). In vitro
(63), 8771–8774. doi:10.1039/c4cc03491k reconstitution of the cyclosporine specific p450 hydroxylases using
Li, P., and Merz, K. M., Jr (2016b). MCPB.py: a Python based metal center heterologous redox partner proteins. J. Ind. Microbiol. Biotechnol. 44 (2),
parameter builder. J. Chem. Inf. Model. 56, 599–604. doi:10.1021/acs.jcim. 161–166. doi:10.1007/s10295-016-1875-y
5b00674 Urlacher, V. B., and Girhard, M. (2019). Cytochrome P450 monooxygenases in
Li, R.-J., Xu, J.-H., Yin, Y.-C., Wirth, N., Ren, J.-M., Zeng, B.-B., et al. (2016a). biotechnology and synthetic biology. Trends Biotechnol. 37, 882–897. doi:10.
Rapid probing of the reactivity of P450 monooxygenases from the CYP116B 1016/j.tibtech.2019.01.001
subfamily using a substrate-based method. New J. Chem. 40, 8928–8934. doi:10. Wang, F., Zhao, J., Li, Q., Yang, J., Li, R., Min, J., et al. (2020). One-pot biocatalytic
1039/C6NJ00809G route from cycloalkanes to α,ω-dicarboxylic acids by designed Escherichia coli
Li, Z., van Beilen, J. B., Duetz, W. A., Schmid, A., de Raadt, A., Griengl, H., et al. consortia. Nat. Commun. 11 (1), 5035. doi:10.1038/s41467-020-18833-7
(2002). Oxidative biotransformations using oxygenases. Curr. Opin. Chem. Biol. Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A., and Case, D. A. (2004).
6 (2), 136–144. doi:10.1016/S1367-5931(02)00296-X Development and testing of a general amber force field. J. Comput. Chem. 25
Maier, J. A., Martinez, C., Kasavajhala, K., Wickstrom, L., Hauser, K. E., and (9), 1157–1174. doi:10.1002/jcc.20035
Simmerling, C. (2015). ff14SB: improving the accuracy of protein side chain and Wang, Z. J., Peck, N. E., Renata, H., and Arnold, F. H. (2014). Cytochrome P450-
backbone parameters from ff99SB. J. Chem. Theory Comput. 11 (8), 3696–3713. catalyzed insertion of carbenoids into N-H bonds. Chem. Sci. 5, 598–601.
doi:10.1021/acs.jctc.5b00255 doi:10.1039/C3SC52535J
Mallinson, S. J. B., Machovina, M. M., Silveira, R. L., Garcia-Borràs, M., Gallup, N., Xia, Y., Li, K., Li, J., Wang, T., Gu, L., and Xun, L. (2019). T5 exonuclease-
Johnson, C. W., et al. (2018). A promiscuous cytochrome P450 aromatic dependent assembly offers a low-cost method for efficient cloning and site-
O-demethylase for lignin bioconversion. Nat. Commun. 9 (1), 2487. doi:10. directed mutagenesis. Nucleic Acids Res. 47 (3), e15. doi:10.1093/nar/gky1169
1038/s41467-018-04878-2 Zhang, A., Zhang, T., Hall, E. A., Hutchinson, S., Cryle, M. J., Wong, L. L., et al.
Mulo, P., and Medina, M. (2017). Interaction and electron transfer between (2015). The crystal structure of the versatile cytochrome P450 enzyme CYP109B1
ferredoxin-NADP+ oxidoreductase and its partners: structural, functional, from Bacillus subtilis. Mol. Biosyst. 11 (3), 869–881. doi:10.1039/C4MB00665H
and physiological implications. Photosynth. Res. 134, 265–280. doi:10.1007/ Zhang, X., Peng, Y., Zhao, J., Li, Q., Yu, X., Acevedo-Rocha, C. G., et al. (2020a).
s11120-017-0372-0 Bacterial cytochrome p450-catalyzed regio- and stereoselective steroid
Munro, A. W., Girvan, H. M., and McLean, K. J. (2007). Variations on a (t) hydroxylation enabled by directed evolution and rational design. Bioresour.
heme--novel mechanisms, redox partners and catalytic functions in the Bioprocess. 7 (1). doi:10.1186/s40643-019-0290-4
cytochrome P450 superfamily. Nat. Prod. Rep. 24 (3), 585–609. doi:10. Zhang, Z., Li, Q., Wang, F., Li, R., Yu, X., Kang, L., et al. (2020b). One-pot
1039/B604190F biosynthesis of 1,6-hexanediol from cyclohexane by de novo designed cascade
Nelson, D. R. (2018). Cytochrome P450 diversity in the tree of life. Biochim. biocatalysis. Green Chem. 22 (21), 7476–7483. doi:10.1039/D0GC02600J
Biophys. Acta 1866 (1), 141–154. doi:10.1016/j.bbapap.2017.05.003
Omura, T., and Sato, R. (1964). The carbon monoxide-binding pigment OF liver Conflict of Interest: The authors declare that the research was conducted in the
microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239 (7), absence of any commercial or financial relationships that could be construed as a
2370–2378. doi:10.1016/s0021-9258(20)82244-3 potential conflict of interest.
Plas, J. V. D., Groot, R. D., Woortman, M., Cremers, F., and Weisbeek, P. (1988).
Genes encoding ferredoxins from anabaena sp. pcc 7937 and synechococcus sp. Copyright © 2021 Zhang, Hu, Peng, Gao, Xing, Wang and Li. This is an open-access
pcc 7942: structure and regulation. Photosynth. Res. 18 (1-2), 179–204. doi:10. article distributed under the terms of the Creative Commons Attribution License (CC
1007/BF00042984 BY). The use, distribution or reproduction in other forums is permitted, provided the
Prier, C. K., Zhang, R. K., Buller, A. R., Brinkmann-Chen, S., and Arnold, F. H. original author(s) and the copyright owner(s) are credited and that the original
(2017). Enantioselective, intermolecular benzylic C-H amination catalysed by publication in this journal is cited, in accordance with accepted academic practice.
an engineered iron-haem enzyme. Nature Chem. 9 (7), 629. doi:10.1038/nchem. No use, distribution or reproduction is permitted which does not comply with
2783 these terms.
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